Recent genome wide association studies suggest that genes whose function mediates and modulates neural circuits affect a wide variety of behaviors ranging from social behaviors to focused repetitive behaviors. However the causal basis for these associations is not clear.
The Cappecchi lab has identified a mouse where altered microglia function, the immune system of the brain, appears causal for a distinct behavioral change. Further, a bone marrow transplant prevents this repetitive behavior. In the mouse, alteration in the immune system (microglia) is directly linked with behavioral change. Mice with a mutation in the Hoxb8 gene show unexpected behavior manifested by repetitive grooming behavior.
There are two principle sources of microglia in mammals: a resident population that is present in the brain early during embryogenesis prior to vascularization, and a second population derived from bone marrow that enters the brain after birth. Hoxb8 exclusively labels the bone marrow population. Having demonstrated a direct relationship between altered microglia and a behavioral change, how microglia interacts with neural circuits and affects behavior can be determined. Further, how alteration in microglial function induces changes in neural circuit function and leads to distinct behavioral change in awake behaving mice can be determined.
Conventional technology has been unable to precisely measure the fate of microglia or neurons that are activated by optogenetic stimulations. The most common imaging modality for a live mouse brain is two-photon confocal fluorescence microscopy. In two-photon confocal fluorescence microscopy, a femtosecond laser is tightly focused through the brain tissue. Due to the high intensities at the focus, two-photon fluorescence is induced at the focus and the signal photons are collected. Two-photon confocal fluorescence microscopy allows for penetrating into the brain tissue up to ˜1 mm. Resolution in the order of a few micrometers has been demonstrated. Unfortunately, this technique typically involves an expensive and complicated equipment setup. Two-photon confocal fluorescence microscopy further involves scanning the focal spot, which reduces temporal resolution. The imaging speed can be increased at higher excitation power, but this also typically causes damage in living tissue.
In conventional optical microscopy (including 2-photon microscopy), resolution is limited by diffraction to about half the excitation wavelength. In the case of 2-photon microscopy, this excitation wavelength is half of the laser wavelength, which is typically in the infrared. Therefore, spatial resolution is still limited. Recently, several methods have allowed production of images which are not diffraction limited in the far field. A family of methods called Reversible-Saturable-Optical-Fluorescence Transitions (RESLOFT) use the transition between bright and dark states to selectively illuminate the sample in a region smaller than the diffraction limit.
Saturated-Structured-Illumination Microscopy (SSIM9) and Saturated-Pattern-Excitation Microscopy (SPEM10) use Moire fringes to move higher spatial frequencies into the optically resolvable region limited by diffraction. These Moire fringes are generated by the product of the local density of fluorescent probes attached to the sample and the excitation light. While a similar method using linear illumination microscopy is capable of improving the resolution by a factor of two, an emission rate, which depends nonlinearly on the illumination intensity can produce a theoretical unlimited resolution. Such emission rate is obtained by illuminating the sample with a sinusoidal pattern with peak intensity that is higher than the emission rate of the fluorophore. The result is emission with a non-sinusoidal rate, which contains higher spatial frequencies than the illumination pattern itself.
A different approach is taken in the related approaches termed Stochastic-Optical-Reconstruction Microscopy (STORM) and Photoactivation-Localization Microscopy (PALM). In these methods, photoactivatable molecules are used to perform time-multiplexed superresolution. A pump laser beam illuminates the fluorescent molecules. This pump illumination statistically activates some of the molecules into a fluorescing state. Since the active molecules are well separated (i.e., they are spatially sparse compared to the diffraction limit), they can be localized by Gaussian fitting. The molecules are then photobleached and another cycle of activation-localization-photobleaching is performed. With a sufficient number of such cycles, every fluorophore in a given field of view can be probed. Finally, the localization data is used to reconstruct a super-resolved image. Although this technique is widefield, a large number of frames are used for high-resolution imaging, which makes this technique slow. Furthermore, in its conventional implementation, only sliced brain tissue can be imaged since a full confocal microscope is used.
FIGS. 1A-1C illustrate some example conventional imaging technologies. FIG. 1A illustrates Stimulated Emission Depletion (STED) microscopy. A focused spot excites fluorescence. A ring-shaped beam quenches fluorescence from everywhere except its center. The signal is then collected from a sub-diffraction-limited region at the center of the second beam. FIG. 1B shows a comparison of a confocal image (background) and a STED image (inset overlay) of the mitochondrial inner membrane. FIG. 1C illustrates a schematic of Photoactivation Localization Microscopy (PALM) techniques. A PALM image is comprised of multiple frames taken at different instances of time that are post-processed into a single image.
Computational imaging through a multi-mode fiber has been used previously. In this case, light from the object is scrambled due to multiple spatial modes in the optical fiber. Complicated interferometer-based calibration systems coupled with speckle imaging methods were used to reconstruct the object details computationally. Unfortunately, the resolution of such an approach is limited and extension to sub-wavelength dimensions is not readily feasible. Related to this method, other groups have focused a spot through multi-mode fibers and scanned the spot to create images. However, these approaches are limited in speed, field of view and resolution. Furthermore, all these techniques tend to require fairly complex and sensitive hardware, at least for the calibration process.